Dataset Information

Duplex Electrochemical DNA Sensor to Detect Bacillus anthracis CAP and PAG DNA Targets Based on the Incorporation of Tailed Primers and Ferrocene-Labeled dATP.

ABSTRACT: We report the duplex amplification of two plasmid DNA markers involved in the virulence of Bacillus anthracis, CAP and PAG, and the direct electrochemical detection of these amplicons. The method consists of the simultaneous amplification of the two targets in a single-pot reaction via polymerase chain reaction (PCR) using tailed primers and ferrocene-labeled dATP. Following amplification, the PCR products hybridize to probes immobilized on electrodes in a microfabricated electrode array chip. The incorporated ferrocene labeled dATP is then detected using square wave voltammetry. We evaluated the effect of electrolyte cations, anions, and concentration to condense, bend, and shrink double-stranded DNA and their effect on the intensity of the ferrocene signal. We obtained detection limits of 0.8 and 3.4 fM for CAP and PAG targets, respectively. We successfully developed a method to detect the presence of both targets in genomic DNA extracted from real samples.

SUBMITTER: Magrina I

PROVIDER: S-EPMC6933787 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Publications

Duplex Electrochemical DNA Sensor to Detect <i>Bacillus anthracis</i> CAP and PAG DNA Targets Based on the Incorporation of Tailed Primers and Ferrocene-Labeled dATP.

Magriñá Ivan I Jauset-Rubio Miriam M Ortiz Mayreli M Tomaso Herbert H Simonova Anna A Hocek Michal M O'Sullivan Ciara K CK

ACS omega 20191211 26

We report the duplex amplification of two plasmid DNA markers involved in the virulence of <i>Bacillus anthracis</i>, CAP and PAG, and the direct electrochemical detection of these amplicons. The method consists of the simultaneous amplification of the two targets in a single-pot reaction via polymerase chain reaction (PCR) using tailed primers and ferrocene-labeled dATP. Following amplification, the PCR products hybridize to probes immobilized on electrodes in a microfabricated electrode array ...[more]

PMID: 31891068

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Project description:Non-specific tail sequences are often added to the 5'-terminus of primers to improve the robustness and overall performance of diagnostic assays. Despite the widespread use of tailed primers, the underlying working mechanism is not well understood. To address this problem, we conducted a detailed in vitro and in silico analysis of the enhancing effect of primer tailing on 2 well-established foot-and-mouth disease virus (FMDV) RT-qPCR assays using an FMDV reference panel. Tailing of the panFMDV-5UTR primers mainly affected the shape of the amplification curves. Modelling of the raw fluorescence data suggested a reduction of the amplification efficiency due to the accumulation of inhibitors. In depth analysis of PCR products indeed revealed the rapid accumulation of forward-primer derived artefacts. More importantly, tailing of the forward primer delayed artefacts formation and concomitantly restored the sigmoidal shape of the amplification curves. Our analysis also showed that primer tailing can alter utilisation patterns of degenerate primers and increase the number of primer variants that are able to participate in the reaction. The impact of tailed primers was less pronounced in the panFMDV-3D assay with only 5 out of 50 isolates showing a clear shift in Cq values. Sequence analysis of the target region of these 5 isolates revealed several mutations in the inter-primer region that extend an existing hairpin structure immediately downstream of the forward primer binding site. Stabilisation of the forward primer with either a tail sequence or cationic spermine units restored the sensitivity of the assay, which suggests that the enhancing effect in the panFMDV-3D assay is due to a more efficient extension of the forward primer. ur results show that primer tailing can alter amplification through various mechanisms that are determined by both the assay and target region. These findings expand our understanding of primer tailing and should enable a more targeted and efficient use of tailed primers.

Dataset Information

Duplex Electrochemical DNA Sensor to Detect Bacillus anthracis CAP and PAG DNA Targets Based on the Incorporation of Tailed Primers and Ferrocene-Labeled dATP.

Publications

Duplex Electrochemical DNA Sensor to Detect <i>Bacillus anthracis</i> CAP and PAG DNA Targets Based on the Incorporation of Tailed Primers and Ferrocene-Labeled dATP.

Similar Datasets

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